AU2016209724A1

AU2016209724A1 - Nanocarrier delivery system for hydrophobic substances

Info

Publication number: AU2016209724A1
Application number: AU2016209724A
Authority: AU
Inventors: Ju Yen FU; Kalanithi Nesaretnam; Doryn Meam Yee Tan
Original assignee: Palm Oil Research and Development Board
Current assignee: Palm Oil Research and Development Board
Priority date: 2015-01-23
Filing date: 2016-01-21
Publication date: 2017-09-14
Anticipated expiration: 2036-01-21
Also published as: GB201713495D0; CN107427471B; WO2016118001A1; CN107427471A; MY197126A; GB2551453A; AU2016209724B2; GB2551453B

Abstract

A nanocarrier delivery system for hydrophobic substances comprising a composition of nanoparticles. The nanoparticle comprises a surfactant of ascorbyl palmitate, a cholesterol, a hydrophilic polymer, a functionalized polymer containing a carboxylic end group, a chemical linker and a targeting protein. A method of producing the nanoparticles and the use of the nanoparticle for the manufacture of a pharmaceutical in the treatment and/or prevention of cancer and cancer related conditions.

Description

WO 2016/118001 PCT/MY2016/050004 1

NANOCARRIER DELIVERY SYSTEM FOR HYDROPHOBIC SUBSTANCES

FIELD OF INVENTION

This invention generally relates to nanocarrier delivery systems. More particularly the invention relates to nanoparticles particularly niosomes for encapsulating hydrophobic substances such as tocotrienol, the method for producing the nanocarrier and the resulting pharmaceutical used in the treatment of cancer and cancer related conditions.

BACKGROUND ART

Nanoparticle research is currently an area of intense scientific interest due to a wide variety of potential applications in biomedical, optical and electronic fields. A bulk material should have constant physical properties regardless of its size, but at the nanoscale size-dependent properties are often observed. Thus, the properties of materials change as their size approaches the nanoscale. These properties are advantageous in many industrial applications. One of the advantageous property of nanoparticles is its ability to suspend in fluids since the interaction of the particle surface with the solvent is strong enough to overcome density differences, which otherwise usually result in a material either sinking or floating in a liquid.

Nanoparticles can be linked to biological molecules that can act as address tags, to direct the nanoparticles to specific sites within the body. Liposomes and niosomes are used for such purposes. A liposome is an artificially-prepared spherical vesicle composed of a lamellar phase lipid bilayer whereas a niosome is a non-ionic surfactant based spherical vesicle formed mostly by non-ionic surfactant and cholesterol incorporation as an excipient. Niosomes are structurally similar to liposomes in having a bilayer, however, the materials used to prepare niosomes make them more stable and thus niosomes offer many more advantages over liposomes. Niosomes can improve the therapeutic performance and increases the bioavailability of the molecules by protecting the molecule from the biological environment, resulting in better availability and controlled drug delivery by restricting the drug effects to targeted cells. PCT/MY2016/050004 WO 2016/118001 2

One compound that is considered for targeted delivery is γ-tocotrienol. Tocotrienols are members of the vitamin E family, an essential nutrient for the body that acts as an antioxidant. Tocotrienols are natural compounds found in select vegetable oils, including rice bran oil and palm oil, wheat germ, barley, saw palmetto, anatto, and certain other types of seeds, nuts, grains, and the oils derived from them. Palm oil contains particularly high concentrations of tocotrienol (up to 70% in its vitamin E content) compared to the rest. Tocotrienol is an effective antioxidant because its unsaturated side chain facilitates penetration into saturated fatty layers of the brain and liver. This may aid in the protection against stroke and neurodegeneration diseases. Tocotrienols can also lower tumor formation, DNA damage and cell damage as well as lowering cholesterol.

One of the major obstacles in formulating pharmaceutical compositions of tocotrienol is its high hydrophobicity. Low aqueous solubility limits the efficacy of a drug, limiting its clinical application. Conventional techniques used to increase the solubility utilizes organic solvents, surfactants, microemulsions etc. However, these methods are not always effective and require very high excipient to drug ratios of at least 15:1, and up to 1000:1, which may result in undesired side effects due to the high concentrations of excipient, compared to the drug itself.

There are several methods of improving the bioavailability of hydrophobic substances using nanoparticles. PCT publication no. WO 2011/028757 A1 discloses a tocotrienol composition that comprises a group of particles having a diameter of about 250-1000 nm where the particles consist of tocotrienol, a non-tocotrienol lipid and a surface active agent. The composition is used as a pharmaceutical composition together with a statin. The statin and tocotrienol is encapsulated in a nanostructured lipid carrier. The nanostructured lipid carrier increases the absorption rate of tocotrienol into the tissue as compared to the absorption rate of tocotrienol without the non-tocotrienol lipid. However, the particle does not contain a targeting molecule for targeted delivery of the tocotrienol. Additionally the particle size of the composition of the PCT publication is 250-1000 nm which is significantly larger than the IUPAC definition of a nanoparticle. Larger particle sizes will result in different properties of the particles itself. At larger sizes, the particle also loses a nanoparticle's ability to suspend in fluids which will result in the particles separating from the suspension fluid. WO 2016/118001 PCT/MY2016/050004 3

Two journal publications published by one of the inventors, Fu, et al., 20091 and Fu, et al., 20112 discloses methods of encapsulating tocotrienol in niosomes that were conjugated with transferrin protein molecules for targeted delivery to tumour cells. The formulations use Span 60 as a major surfactant in the manufacture of the niosomes. In the 2011 publication, the particle size of the conjugated particle was determined to be 341 nm, well above the size limit nanoparticle defined by IUPAC whereas the particle size in the 2009 publication was determined to be 137 nm. It was discovered that the formulations in the 2009 and 2011 publication were unstable; up to 30% of the particles prematurely released the encapsulated tocotrienol. The formulations also were found to cake and agglomerate when freeze-dried, raising problems of manufacturing the formulation into a pharmaceutical composition.

Flence, there is a need for a nanocarrier delivery system that delivers hydrophobic substances to a targeted cell that is stable and non-toxic that is suitable to be used to produce a pharmaceutical composition. This invention thus aims to alleviate some or all of the problems of the prior art. 1 Fu, J. Y., Blatchford, D. R., Tetley, L. & Dufes, C., 2009. Tumor regression after systemic administration of tocotrienol entrapped in tumor-targeted niosomes. Journal of Controlled Release, Issue 140, pp. 95-99. 2 Fu, J. Y. et al., 2011. Novel tocotrienol-entrapping niosomes can eradicate solid tumors after intravenous administration. Journal of Controlled Release, Issue 154, pp. 20-23.

SUMMARY OF THE INVENTION

According to an aspect of the invention, there is provided a nanocarrier delivery system for hydrophobic substances comprising a composition of nanoparticles. The nanoparticle comprises the following components: a surfactant comprising ascorbyl palmitate; a cholesterol; a hydrophilic polymer; a functionalized polymer containing a carboxylic end group; a chemical linker; and a targeting protein. PCT/M Y2016/050004 WO 2016/118001 4

The targeting protein forms targeted-nanoparticles for enhanced targeted delivery of the hydrophobic substances in the body of a mammal and the nanoparticles has improved storage stability with degradation of less than about 5%. The nanoparticle is a niosome that is substantially spherical in shape. The niosome has a particle size of less than 150 nm.

In an embodiment, the ascorbyl palmitate has a molar ratio of 0.5 to 1.00 of the nanoparticle composition.

In a further embodiment, the cholesterol is 3p-hydroxy-5-cholestene, 5-cholesten-3p-ol. The molar ratio of cholesterol in the nanoparticle composition is 0.5 to 1.00.

In another embodiment, the hydrophilic polymer is vitamin E TPGS (TPGS). The molar ratio of TPGS is 0.1 to 0.2 of the nanoparticle composition.

In an embodiment, the functionalized polymer is DSPE-PEG(2000) carboxylic acid. The molar ratio of DSPE-PEG(2000) carboxylic acid is 0.02 to 0.06 of the nanoparticle composition.

In another embodiment, the chemical linker is l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and/or sulfo-N-hydroxysuccinimide (Sulfo-NHS). The molar concentration of the chemical linker is 0.09 M of the nanoparticle composition.

In a further embodiment, the targeting protein is transferrin. The molar concentration of transferrin is 60 mg/ml of the nanoparticle composition.

In a second aspect of the invention, there is provided a method of producing the nanocarrier delivery system where the method comprises the following steps: i) dissolving a hydrophobic substance in an organic solvent to form a hydrophobic solution; ii) mixing the hydrophobic solution with surfactant, cholesterol and hydrophilic polymer to produce an unpurified solution; iii) removing excess organic solvents from the unpurified solution to form a dry film; iv) hydrating the dry film into a suspension; PCT/MY2016/050004 WO 2016/118001 5 v) separately providing the chemical linkers in a buffer solution to produce a buffer suspension; vi) mixing the unpurified niosomes into the buffer suspension; vii) subsequently adding a targeting protein solution; viii) purifying the resulting mixture; and ix) freeze drying the resulting mixture.

In an embodiment, the organic solvent is hexane and methanol. The organic solvent of hexane and methanol added has a ratio of 9:1.

In a further embodiment, the hydrating step in step (iv) uses phosphate buffered saline (PBS).

In another embodiment, the buffer solution used in step (v) is 2-ethanesulfonic acid (MES).

In a separate embodiment, the targeting protein solution comprises transferrin and PBS.

One hydrophobic substance that may be encapsulated in the nanocarrier delivery system of this invention is y-tocotrienol.

In another aspect of the invention, the nanocarrier delivery system is used in the manufacture of a pharmaceutical for administration to a mammal in the treatment of cancer and cancer related conditions. The pharmaceutical may be administered orally or intravenously at an effective dose of about 5 mg/kg to about 500 mg/kg body weight with a period of treatment of 10 to 60 days.

It is an aim of the present invention to provide a nanocarrier delivery system for encapsulating hydrophobic substances, though not exclusively, for encapsulating y-tocotrienol used for the manufacture of a pharmaceutical composition.

The nanocarrier delivery system and resulting pharmaceutical of this invention provides for various advantages which will be further elaborated in the following pages. WO 2016/118001 PCT/MY2016/050004 6

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated, although not limited, by the following description of embodiments made with reference to the accompanying drawings in which: FIGURE 1 illustrates a graph showing the percentage of tocotrienol retained in DSPE-PEG-niosomes and transferrin-conjugated niosomes at different time points upon storage at 3-5°C. Results are mean values of triplicate analyses ± SD. FIGURE 2 illustrates a chromatogram of tocotrienol and PMC extracted from nanoparticles. FIGURE 3 illustrates a graph showing the percentage of tocotrienol released from Span 60 niosomes stored at 4°C over 30 days which was measured in triplicate.

DETAILED DESCRIPTION OF THE EMBODIMENTS

This invention is directed to a nanocarrier delivery system comprising nanoparticles particularly niosomes for encapsulating hydrophobic substances such as tocotrienol, methods for producing the nanocarrier and the resulting pharmaceutical composition.

Nanoparticle Composition

The nanoparticle of this invention mainly comprises a surfactant, a cholesterol, a hydrophilic polymer, a functionalized polymer containing a carboxylic end group, a chemical linker, a targeting protein and a hydrophobic substance.

Any suitable surfactant may be used. Preferably, ascorbyl palmitate (AP) is used as the surfactant. AP is added to the nanoparticle composition at a molar ratio of 0.5 to 1.00. Preferably the molar ratio is 1.00. AP is an analogue of vitamin C, biodegradable and can be fully metabolised by the human body, thus minimizing toxic effects. AP is also biocompatible and has minimum inflammatory and immunological response when administered to humans. PCT/MY2016/050004 WO 2016/118001 7

Any suitable cholesterol may be used. In the present invention, 33-hydroxy-5-cholestene, 5-cholesten-3p-ol was used as the cholesterol component. The cholesterol component is added to the nanoparticle composition at a molar ratio of 0.5 to 1.00. Preferably, the molar ratio is 0.5.

Cholesterol is a major component in the manufacture of the nanoparticle as the cholesterol and surfactant component align to form the lipid bilayer characteristic of a niosome. As niosomes are non-ionic as opposed to structurally similar liposomes, niosomes are more chemically stable and have longer storage life compared to liposomes which offer more advantages.

Any suitable hydrophilic polymer may be used. In the present invention, D-a-Tocopherol polyethylene glycol 1000 succinate, also known as vitamin E TPGS or simply TPGS was used as the hydrophilic polymer component. TPGS is added to the nanoparticle composition at a molar ratio of 0.1 to 0.2. Preferably, the molar ratio is 0.2.

Any suitable functionalized polymer may be used. In the present invention, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000], also known as DSPE-PEG(2000) carboxylic acid was used as the functionalized polymer component. DSPE-PEG(2000) carboxylic acid is added to the nanoparticle composition at a molar ratio of 0.02 to 0.06. Preferably, the molar ratio is 0.04.

The hydrophilic polymer and functionalized polymer act as co-surfactants in the formation of the nanoparticles. The surfactant molecules - AP, TPGS and DSPE-PEG(2000), tend to orientate themselves in such a way that the hydrophilic ends of the non-ionic surfactant point outwards, while the hydrophobic ends face each other to form the bilayer. Since both TPGS and DSPE-PEG(2000) are non-ionic molecules, together with AP and the cholesterol, they form a non-ionic niosome. Niosomes have high compatibility with biological systems and low toxicity because of their non-ionic nature.

Chemical linkers are crosslinkers for covalent conjugation between the functionalised polymer and the targeting protein. The covalent bond ensures sufficient stability of the conjugates. PCT/MY2016/050004 WO 2016/118001 8

Any suitable chemical linker may be used. In the present invention, l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and sulfo-N-hydroxysuccinimide (Sulfo-NHS) were used as the chemical linker component. The chemical linker component is added to the nanoparticle composition at a molar concentration of 0.09 M.

The carboxylates on functionalized polymer reacts with EDC to form unstable esters. NHS is added to increase the stability and solubility of this esters intermediate, which will react with the amine group on the targeting protein. This will be elaborated in the following paragraph.

Any suitable targeting protein may be used. In the present invention, transferrin was used as the targeting protein component. Transferrin is added to the nanoparticle composition at a molar concentration of 60 mg/ml.

Transferrin is a protein that transports iron in the body of a mammal that specifically identifies their receptors i.e. transferrin receptors. Transferrin receptors are often overexpressed in cancer cells due to the high demand of iron for tumour growth. Hence, transferrin may be used as a targeting strategy for specifically delivering active ingredients to cancer cells by conjugating transferrin to the molecule that will deliver the active ingredient to the cell.

The nanocarrier of this invention may encapsulate any suitable hydrophobic substance. An example is γ-tocotrienol (tocotrienol). The tocotrienol may be added to the nanoparticle composition at a molar ratio of 0.24 M.

The targeting protein is conjugated onto the niosomes which will act as an address tag that will direct the niosomes to a specific cell in the body such as a cancer cell. Due to the tocotrienol being encapsulated in the niosomes, the tocotrienol is protected from the biological environment while in the blood stream, resulting in better bioavailability and targeted delivery by restricting the drug effects to specific cells in the body.

The nanoparticle formulation of the present invention may also be used to encapsulate hydrophilic molecules instead of hydrophobic molecules. Niosomes can entrap both hydrophobic drugs into vesicular bilayer membranes and hydrophilic drugs in the aqueous compartment in the center of the niosome. WO 2016/118001 PCT/MY2016/050004 9

It was surprisingly discovered that the nanoparticle formulation is significantly more stable in comparison to the two previous formulations published in Fu, et al., 2009 and Fu, et al., 2011. In the 2009 and 2011 publications, the formulations suffered up to 30% premature release of tocotrienol after storage for 30 days at 4°C. The test results for the 2009 and 2011 publication formulations are shown in Example 8.

The formulation of the present invention was found to remain stable even after 2 months of storage at 4°C with less than 5% degradation. This is shown in Example 6.

Method of Producing the Nanoparticle Composition

The method of producing the nanoparticle composition mainly comprises the following steps: i) dissolving a hydrophobic substance in an organic solvent to form a hydrophobic solution; ii) mixing the hydrophobic solution with the surfactant, the cholesterol and the hydrophilic polymer to produce an unpurified solution; iii) removing excess organic solvents from the unpurified solution to form a dry film; iv) hydrating the dry film into a suspension; v) separately providing the chemical linkers in a buffer solution to produce a buffer suspension; vi) mixing the unpurified niosomes into the buffer suspension; vii) subsequently adding a targeting protein solution; viii) purifying the resulting mixture; and ix) freeze drying the resulting mixture.

Any suitable organic solvent may be used in step (i). Preferably, hexane and methanol is used as the surfactant. The ration of hexane to methanol is 9:1 by volume. The hydrophobic substance and organic solvent are mixed until homogeneous to produce a hydrophobic solution.

The surfactant, cholesterol and hydrophilic polymer are then added to the hydrophobic solvent in step (ii) and mixed until homogenous to produce an unpurified solution. WO 2016/118001 PCT/MY2016/050004 10

Any suitable process for removing excess solvent from the unpurified solution may be used in step (iii). For example, a rotary evaporator may be used to remove the excess solvent. When the excess solvent is removed, a thin, dry film forms on the walls of the flask of the rotary evaporator.

In step (iv), any method of hydrating may be used. In the present invention, phosphate buffered saline (PBS) may be used to hydrate the dry film into a suspension at room temperature followed by probe sonification for 4 minutes at 80% of the maximum output.

In step (v), a buffer suspension is separately provided by mixing the chemical linkers and buffer solution. Any suitable buffer solution may be used. For example, 2-ethanesulfonic acid (MES) may be used as the buffer solution. The chemical linkers and buffer solution are mixed until homogeneous to produce a buffer suspension.

The stored unpurified niosomes from step (vi) are then added to the buffer suspension and incubated at room temperature for 15 minutes while gently mixing until homogeneous.

This step is followed by adding a targeting protein solution. Prior to adding the targeting protein solution, the mixture of step (vii) is adjusted to a pH of 7-8. Any suitable pH adjuster may be used. For example, sodium hydroxide was used to adjust the pH level in the present invention. A targeting protein solution comprises a targeting protein and a solvent. Any suitable targeting protein may be used. For example, transferrin was used as the targeting protein in the present invention. The targeting protein was dissolved in phosphate buffer solution (PBS) to obtain the targeting protein solution. The resulting solution is mixed by stirring for 3 hours at room temperature until homogenous for conjugation to take place.

The resulting mixture is then purified to remove any unbound materials. Any suitable purification method may be used. For example, ultracentrifugation at 41,000 rpm with operating temperature of 4°C for 3 hours was used to purify the resulting mixture to yield only purified transferrin-conjugated niosomes. WO 2016/118001 PCT/MY2016/050004 11

Once the resulting mixture of purified, the mixture is then freeze-dried. Any suitable freeze-drying method may be used. For example, a Freezone Labconco freeze drying system may be used. The mixture is freeze-dried at -84°C for 24 hours to produce a stable powder that is suitable for storage and subsequent use for manufacturing a pharmaceutical.

Use of the freeze-dried niosomes

The present invention provides a novel use of transferrin-conjugated niosomes comprising a therapeutically effective amount of the niosomes. The niosomes are useful in the treatment and/or prevention of cancer or cancer related conditions.

In another embodiment, the nanoparticle composition comprising the niosomes may be processed into a pharmaceutical for administration to a mammal such as a human in the treatment and/or prevention of cancer or cancer related conditions.

The pharmaceutical composition may contain any other suitable additive for facilitating easy consumption/administration of the medicament and also for prolonging the shelf life of the medicament.

Any suitable consumable form may be used. In an embodiment, forms such as pellets, tablets, capsules, granules or liquid may be used.

The pharmaceutical may be packaged in unit doses for facilitating the administration to the patient. Any suitable packaging may be used. For example, blister packs for tablets and capsules, vials for liquid concentrate or liquid suspension, or IV pouches for intravenous therapy.

The medicament may be administered in a fixed unit dosage to effectively treat and/or prevent cancer or cancer related conditions. Any suitable effective dose may be used. Preferably, the effective dose is about 5 to about 500 mg/kg body weight. The management period may be of any suitable length. Preferably, the management period is about 10 to 60 days. WO 2016/118001 PCT/M Y2016/050004 12

EXAMPLE

The following Examples illustrate the various aspects, methods and steps of the process of this invention. These Examples do not limit the invention, the scope of which is set out in the appended claims.

Example 1

Preparation of non-conjugated nanoparticles using film hydration method

Components to construct nanoparticle were identified including a) surfactant, b) cholesterol, c) hydrophilic polymer, d) functionalized polymer containing carboxylic end group, e) chemical linker and f) targeting protein.

The components were prepared based on molar ratios as shown in Table 1 below.

Table 1: The compositions (molar ratio) of formulations prepared.

Formulation Molar ratio ol components AP Cholesterol Vitamin E TPGS DSPE- PEG-(2000)-carboxylic acid A1 1.00 0.50 - - A2 1.00 0.75 - - A3 1.00 1.00 - - A4 0.75 1.00 - - A5 0.50 1.00 - - B1 1.00 0.50 0.10 - B2 1.00 0.50 0.20 - Cl 1.00 0.50 0.20 0.02 C2 1.00 0.50 0.20 0.04 C3 1.00 0.50 0.20 0.06

Gamma tocotrienol is first dissolved in a mixture of organic solvents consisting of hexane and methanol in a round bottom flask.

After that, ascorbyl palmitate (AP), cholesterol and hydrophilic polymer (TPGS) were added into the solvents with varied molar ratio. The solvents were removed by using rotary evaporator at 50°C until the formation of a thin, dry film on the inner wall of the flask. The film is then hydrated with 10 ml of PBS at room temperature followed by probe sonication for 4 minutes with the instrument set at 80% of its maximum output. The purification of niosomes was carried out using ultracentrifugation (2 cycles, 41,000 PCT/MY2016/050004 WO 2016/118001 13 rpm, 4 hours, 25°C). The niosomes pellet was resuspended with 10 ml of PBS after each ultracentrifugation cycle.

The nanosuspensions were freeze dried and harvested as off-white powder. The powder was then stored in desiccators protected from light.

Example 2

Conjugation of transferrin onto nanoparticles

For preparation of transferrin-conjugated niosomes, cross-linking procedures as described in Ishida et al. and Paola et al. were adopted. Firstly, EDC and sulfo-NHS were prepared in MES buffer at 90 pmol/mL. Then, 10 mL of EDC and lOmL of Sulfo-NHS solutions were added into 10 mL of unpurified niosomes. The mixture was incubated at room temperature for 15 minutes with gentle stirring.

After incubation, the pH of the mixture was adjusted to pH 7-8 with 1.0 M NaOH. Then, 2mL of trasnferrin solution (prepared using 120 mg Transferrin in 2.0 mL of PBS) were added to the mixture. The mixture was incubated again for 3 hours with gentle stirring at room temperature for conjugation to take place.

Unbound materials were removed using ultracentrifugation method at 41,000 rpm and 4°C for 3 hours to yield purified transferrin-conjugated niosomes. All preparation of niosomes were subjected to freeze drying process (-84°C; 24 hours) to produce stable powder.

Example 3

Measuring the drug encapsulation efficiency of the transferrin-conjugated nanoparticles

Formulated nanoparticles were optimized based on encapsulation efficiency, size, zeta potential and morphology. Drug encapsulation efficiency is described as the ratio of drug encapsulated inside the niosomes to the total amount of drug added for the niosome preparation while drug loading efficiency is the ratio of mass of drug encapsulated inside WO 2016/118001 PCT/MY2016/050004 14 the niosomes to the mass of total carrier system. Encapsulation efficiency and drug loading of nanocarriers were measured using high performance liquid chromatography (HPLC) quantification method for vitamin E. Size and zeta potential was measured using Zetasizer Nano ZS, a system that has combined techniques for measurement of particles 5 size, zeta potential and molecular weight of proteins. The technique that responsible for particle size analysis quantifying the Brownian motion of particles. Zeta potential is the electric potential difference between the dispersion medium and the stationary liquid layer surrounding the dispersed particle. It provides an estimation of the magnitude of attractive and repulsive forces between the dispersed particles. Therefore, by measuring 10 the zeta potential, stability of dispersion can be predicted. Morphology of nanoparticles were examined using Transmission electron microscopy (TEM). TEM involves the penetration of high-energy electron beam through a thin sample. The electrons interacted with the sample and subsequently formed an image, providing higher resolution than conventional light microscope. 15

The present invention was able to produce niosomes with particle size less than 150 nm and high homogeneity (polydispersity index less than 0.3). The encapsulation efficiency was shown to be dependent on the ratio of AP to cholesterol (as shown in Table 2). 20 As the ratio decreased, encapsulation efficiency reduced dramatically, from 24.67% (formulation Al) to 3.51% (formulation A5). This finding was consistent with results presented in Gopinath et al. (2004), in which the niosomes were synthesized using AP, cholesterol and dicetyl-phosphate. All of the niosomes were anionic, as they have negative zeta potential value. 25

However, when the amount of cholesterol incorporated is higher than AP (formulation A4 and A5), the zeta potential value became less negative. Since formulation Al had the highest encapsulation efficiency, the molar ratio of AP and cholesterol was selected for further inclusion of vitamin E TPGS into the formulations. PCT/MY2016/050004 WO 2016/118001 15

Table 2: Physical characteristics of niosomes with different formulations. Data represents mean ± SD.

Formulation Particle size/d.nm Polydispersity index Zeta potential/ mV Encapsulation efficiency/ % A1 124 ± 9 0.18 ± 0.03 -72 ±9 24.7 ± 1.5 A2 130 ± 1 0.22 ± 0.01 -79 ± 2 22.2 ± 1.4 A3 116 ± 7 0.20 ± 0.03 -72 ±9 14.1 ± 1.0 A4 131 ± 3 0.15 ± 0.03 -62 ± 7 8.0 ± 0.5 A5 135 ± 1 0.15 ± 0.03 -68 ± 1 3.5 ± 0.1

Example 4

Incorporation hydrophilic polymer into the nanoparticle formulation

Incorporation of hydrophilic polymer (TPGS) into niosome formulations resulted in an elevation of encapsulation efficiency, from 24.7% to 42.9% (as shown in Table 3). When the molar ratio of TPGS doubled, the encapsulation efficiency increased as much as 20%. Therefore, the molar ratio of TPGS was fixed at 0.2 for synthesis of targeted niosomes.

Table 3: Physical characteristics of niosomes coated with hydrophilic polymers. Data represents mean ± SD.

Formulation Particle size/d.nm Polydispersity index Zeta potential/ mV Encapsulation efficiency/ % B1 138 ± 3 0.33 ± 0.04 -82 ± 1 22.5 ± 0.9 B2 120 ± 2 0.23± 0.01 -60 ± 1 42.9 ± 3.6

Example 5

Incorporation functionalized polymer into the nanoparticle formulation

According to Table 4, the optimum molar ratio of DSPE-PEG (2000) carboxylic acid required for synthesis of targeted niosomes was formulation C2, with conjugation efficiency of 13.13%, followed by C3 (11.80%) and Cl (7.84%). Thus, particle size was indirectly proportional to the molar ratio of linker lipid. Moreover, targeted niosomes produced by three different formulations have mid-range polydispersity as their polydispersity index fall within 0.08 to 0.7. Note worthily, C2 also has the lowest polydispersity index among the three formulations. The resulting niosome is labelled as DSPE-PEG niosomes. PCT/MY2016/050004 WO 2016/118001 16

Table 4: Physical characteristics of targeted-niosomes. Data represents mean ± SD.

Formulation Particle size /d.nm Polydispersity index Zeta potential/ mV Tf conjugation efficiency/ % Cl 214 ± 8 0.50 ± 0.10 -80 ± 3 7.8 ± 0.5 C2 194 ± 2 0.29 ± 0.01 -78 ±3 13.1 ± 2.1 C3 178 ± 1 0.42 ± 0.01 -75 ± 1 11.8 ± 0.8

From the above results, it can be concluded that the optimum formulation for non-targeted nanocarrier is molar ratio of AP:cholesterol:TPGS is 1.0:0.5:0.2. For targeted niosomes, the optimum molar ratio of AP:cholesterol:TPGS:DSPE-PEG(2000) carboxylic acid is 1.0:0.5:0.2:0.04. The niosomes conforms to a spherical shape under TEM.

Example 6

Stability test of nanoparticle formulation DSPE-PEG niosomes and transferrin-conjugated niosomes powders (24 mg each) were dissolved separately in 12 mL of PBS (2 mg/mL) in a universal bottle. The nanosuspensions were stored in the capped bottles at 3-5°C and protected from light for 2 months. The percentage of tocotrienol retained in the niosomes was used as the parameter to evaluate the stability of niosomes, since instability would be reflected in drug leakage and a decrease in the percentage of drug retained. The niosomes were sampled after storage period of 2 weeks, 1 month and 2 months. The nanosuspensions were observed for colour change and the amount of tocotrienol encapsulated inside the niosomes was determined using the HPLC method. The percentage of tocotrienol retained was calculated from the ratio of the encapsulation efficiency after storage to the initial encapsulation efficiency of the tocotrienol. A new batch of DSPE-PEG niosomes and transferrin-conjugated niosomes were synthesized in order to access the stability of the niosomes when stored at 3-5°C. Immediately after the production of DSPE-PEG niosomes and transferrin-conjugated, the amount of tocotrienol was quantified using HPLC method. The encapsulation efficiencies of DSPE-PEG niosomes and transferrin-conjugated niosomes were 41.35% and 26.82%, respectively. After 2 months of storage at 3-5°C, 96.4% of tocotrienol was retained in DSPE-PEG niosomes while 90.9% of tocotrienol was conserved in transferrin-conjugated niosomes as shown in Figure 1. Both DSPE-PEG niosomes and transferrin-conjugated niosomes remained stable throughout the storage period, since no disparity was observed in the tocotrienol content. Also, neither aggregation nor change in the colour PC T/MY2016/050004 WO 2016/118001 17 of nanosuspesions happened upon 2 months of storage Therefore, it was postulated that tocotrienol was encapsulated within the bilayer membrane of the niosomes, instead of adsorbed on the surface of niosomes.

Example 7

Quantification of tocotrienol using normal phase liquid chromatography

Chromatographic separations were performed on a silica-based analytical column maintained at 25±1°C. The mobile phase comprised of a mixture of n-hexane, 1,4-dioxane and isopropanol (97.5:2:0.5% v/v/v). The injection volume was set at 100 pL, the flow rate was fixed at lmL/min and the detection was set as excitation wavelength at 295 nm and emission wavelength at 325 nm. The mobile phase was degassed by 30 minutes sonication prior to use. A stock standard solution with concentration of 2000 ppm of γ-tocotrienol was prepared in 20 mL mobile phase. Working standard solutions (200, 100, 50, 10, 5, 0.1, 0.05 ppm) were prepared by making appropriate serial dilution of stock standard solution with mobile phase. 2, 2, 5, 7, 8-Pentamethyl-6-hydroxychroman (PMC) was prepared as the internal standard (IS) stock solution by dissolving 20 mg of PMC powder with mobile phase to a final concentration of 2000 ppm. Working IS solutions (100, 10 and 1 ppm) were prepared by serial dilution of the stock IS solution with mobile phase.

Tocotrienol and IS were extracted from nanoparticles according to method formerly reported by Nesaretnam et al. with slight modification. A volume 1.0 mL nanosuspension was added into universal bottle before spiking with 1 ppm of IS. The solution was vortexed for 10 seconds, followed by the addition of 1.0 mL of 0.9% (w/v) NaCI. The mixture was vortexed again for another 10 seconds before adding 1.0 mL of ethanol and 5.0 mL n-hexane. The mixture was then sonicated for one hour. In order to separate the mixture into water and organic phases, the mixture was centrifuged at 2500 rpm for 15 minutes at temperature 4°C. The upper organic phase was transferred to a trident vial and evaporated to dryness with nitrogen gas before reconstituting with 0.5 mL of mobile phase.

Calibration curves were constructed using ten standard solutions and IS over a concentration range of 0.05-200 ppm. The peak area ratios tocotrienol was plotted PCT/M Y2016/050004 WO 2016/118001 18 against the nominal concentrations. Linear regression analysis was performed using the linear equation y = mx, where y is the peak area ratio of tocotrienol, x is the tocotrienol concentration and m is the slope of the calibration curve. Correlation coefficient (R2) was used to measure the linearity of the calibration curves. The concentrations of tocotrienol in the sample were determined according to the formula:

Concentration of tocotrienol (pg/mL) _ Peak area of y-T3 Total sample volume 1

Peak area of 1 pg standard. Volume injected. Sample weight (g)

The limit of detection (LOD) is the lowest concentration of tocotrienol in spiked standard solutions that produces a peak with a signal-to-baseline noise (S/N) ratio of > 3. Limit of quantification (LOQ) was defined as the concentration on the calibration curve at which quantitative results can be reported with a high degree of confidence that producing a peak with S/N ratio of > 10 (184).

The precision and accuracy of the method were evaluated by analyzing the spiked samples of nanosuspension. The intra-day precision and accuracy were estimated by analyzing triplicates of each sample at the same concentration level at three different times in a day. The same procedure was followed for three consecutive days to determine inter-day precision and accuracy. Intra and inter-assay precisions were expressed as the percentage ratio standard deviation (RSD) of the measured concentrations of nanosuspension: RSD =

Standard deviation of concentration Mean concentration x 100%

Accuracy was studied by analysing the nanosuspension and was expressed as percentage recovery of spiked samples. For acceptable intra- and inter-day values, the accuracy should be within ± 10% of the nominal concentrations.

Under the chromatographic conditions described, tocotrienol was well separated from IS, with retention times of approximately 16.1 min and 10.9 min, respectively. The total run time for each sample was 30 minutes. Figure 2 showed the chromatogram of tocotrienol and PMC extracted from nanoparticles. WO 2016/118001 PCT/MY2016/050004 19

As shown in Table 5, the detector responses for tocotrienol and PMC standards extracted from nanoparticles were linear within the range of 0.05 to 200 ppm with R2 > 0.99.The LOD for both tocotrienol and PMC is 0.05 ppm while the LOQ values for tocotrienol and PMC were 0.1 ppm and 0.05 ppm, respectively.

Table 5. Method validation for quantification of γ-Τ3 and PMC

Analyte Detection range (ppm) Linearity (R2 value) LOD (ppm) LOQ (ppm) Tocotrienol 0.05-200 0.99989 0.05 0.1 PMC 0.05-200 0.99979 0.05 0.05 *Data represents mean.

The accuracy and precision data are summarized in Table 6. The recovery values of tocotrienol and PMC were approximately 97.41% and 94.66%, respectively. The RSD values for interday variation for tocotrienol and PMC were 1.41% and 3.23%, respectively. For intra-assay precision, the %RSD for y-T3 is 2.31% while the %RSD for PMC is 3.93%. Since deviation of detected tocotrienol and PMC concentrations is less than 10% of the nominal concentrations, the validated assay is considered accurate, precise and reliable.

Table 6. Interday and intraday precision and accuracy of y-T3 and PMC in HPLC method validation

Analyte Nominal concentration (ppm) Detected concentration (ppm) Recovery (%) Interday RSD (%) Intra-day RSD (%) Tocotrienol 10.05 9.79 ± 0.15 97.41 ± 1.45 1.41 2.31 PMC 9.85 9.51 ± 0,.40 94.66 ± 3.99 3.23 3.93

*Data represents mean ± SD

Example 8

Stability test of nanoparticle formulation based on 2009 and 2011 publications using Span 60

At days 0, 4, 7, 14, 21, and 30, Span 60 niosomes stored at 4°C were centrifuged at 40,000 rpm for 2 hours. Niosomes pellets were redispersed in PBS where tocotrienol-derived fluorescence was measured at Agitation 295 nm and Aemission 325 nm upon vesicle disruption with isopropanol at dilution 1:100. PCT/MY2016/050004 WO 2016/118001 20

The stability of tocotrienol encapsulation in Span 60 niosomes was quantified in order to give a general idea of possible tocotrienol leakage during vesicle storage at 4°C. At day 7, about 10 to 15% of tocotrienol was released from transferrin-conjugated niosomes and control niosomes. Apart from the burst release within the first 7 days, a sustained 5 release was observed over the next 3 weeks. An average release rate of less than 1% per day was recorded for both transferrin-conjugated niosomes and control niosomes. At day 30, the amount of tocotrienol retained in the vesicles was not significantly different between control and transferrin-conjugated niosomes: up to 33.3 ± 4.9% and 23.8 ± 4.5% of tocotrienol was released from control niosomes and transferrin-conjugated 10 niosomes respectively. A graph showing the progress of tocotrienol release from Span 60 niosomes is shown in Figure 3. 15 As shown in the Examples above particularly Example 6 and 8, it is evident that the nanoparticle formulation of the present invention has far greater stability compared to the 2009 and 2011 published formulations as per Example 8 i.e. up to 30% release of tocotrienol after 30 days as compared to less than 5% after 2 months in the present invention as per Example 6. 20

As will be readily apparent to those skilled in the art, the present invention may easily be produced in other specific forms without departing from its scope.

Claims

1. A nanocarrier delivery system for hydrophobic substances comprising a composition of nanoparticles, said nanoparticle comprising the following components: a surfactant comprising ascorbyl palmitate; a cholesterol; a hydrophilic polymer; a functionalized polymer containing a carboxylic end group; a chemical linker; and a targeting protein, wherein said targeting protein forms targeted-nanoparticles for enhanced targeted delivery of hydrophobic substances in the body of a mammal; and wherein said nanoparticles has improved storage stability with degradation of less than about 5%.

2. The nanocarrier delivery system of claim 1 wherein said nanoparticle is a niosome.

3. The nanocarrier delivery system of claims 1 and 2 wherein said niosome is substantially spherical in shape.

4. The nanocarrier delivery system according to any one of claims 1 to 3 wherein said niosome has a particle size of less than about 150 nm.

5. The nanocarrier delivery system claim 1 wherein the molar ratio of said ascorbyl palmitate is about 0.5 to about 1.00 of the nanoparticle composition.

6. The nanocarrier delivery system according to any one of claims 1 to 5 wherein said cholesterol is 33-hydroxy-5-cholestene, 5-cholesten-33-ol.

7. The nanocarrier delivery system of claim 6 wherein the molar ratio of said cholesterol is about 0.5 to about 1.00 of the nanoparticle composition.

8. The nanocarrier delivery system according to any one of claims 1 to 8 wherein said hydrophilic polymer is vitamin E TPGS (TPGS).

9. The nanocarrier delivery system of claim 8 wherein the molar ratio of said TPGS is about 0.1 to about 0.2 of the nanoparticle composition.

10. The nanocarrier delivery system according to any one of claim 1 to 9 wherein said functionalized polymer is DSPE-PEG(2000) carboxylic acid.

11. The nanocarrier delivery system of claim 10 wherein the molar ratio of said DSPE-PEG(2000) carboxylic acid is about 0.02 to about 0.06 of the nanoparticle composition.

12. The nanocarrier delivery system according to any one of claims 1 to 11 wherein said chemical linker is l-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) and/or sulfo-N-hydroxysuccinimide (Sulfo-NHS).

13. The nanocarrier delivery system of claim 12 wherein the molar concentration of said chemical linker is about 0.09 M of the nanoparticle composition.

14. The nanocarrier delivery system according to any one of claims 1 to 13 wherein said targeting protein is transferrin.

15. The nanocarrier delivery system of claim 14 wherein the molar concentration of said tranferrin is about 60 mg/ml of the nanoparticle composition.

16. A method of producing the nanocarrier delivery system according to any one of the preceding claims, said method comprising the following steps: i) dissolving a hydrophobic substance in an organic solvent to form a hydrophobic solution; ii) mixing said hydrophobic solution with said surfactant, said cholesterol and said hydrophilic polymer to produce an unpurified solution; iii) removing excess organic solvents from said unpurified solution to form a dry film; iv) hydrating said dry film into a suspension; v) separately providing said chemical linkers in a buffer solution to produce a buffer suspension; vi) mixing said unpurified niosomes into said buffer suspension; vii) subsequently adding a targeting protein solution; viii) purifying the resulting mixture; and ix) freeze drying said resulting mixture, wherein said resulting mixture is said nanoparticle composition of claim 1.

17. The method according to claim 16 wherein said organic solvent is hexane and methanol.

18. The method according to claim 17 wherein said organic solvent of hexane and methanol is added in a ratio of about 9:1 by volume.

19. The method according to any one of claims 16 to 18, wherein phosphate buffered saline (PBS) is used in said hydrating step of step (iv).

20. The method according to any one of claims 16 to 19, wherein said buffer solution is 2-ethanesulfonic acid (MES).

21. The method according to any one of claims 16 to 20, wherein said targeting protein solution comprises transferrin and PBS.

22. The nanocarrier delivery system according to any one of the preceding claims, wherein said hydrophobic substance is y-tocotrienol.

23. The nanocarrier delivery system according to any one of the preceding claims, wherein said mammal is a human.

24. Use of the nanocarrier delivery system according to any one of the preceding claims in the manufacture of a pharmaceutical for administration to a mammal in the treatment of cancer and cancer related conditions.

25. The use of claim 24, wherein said pharmaceutical is administered orally or intravenously.

26. The use of claims 24 or 25, wherein said pharmaceutical is administered at an effective dose of about 5 mg/kg to about 500 mg/kg body weight.

27. The use of any one of claims 24 to 26, wherein the period of treatment of said pharmaceutical is about 10 days to about 60 days.